1. Field of the Invention
The present invention relates to a method for preparing a GM1 gangliosidosis human cell model by using the induced pluripotent stem cells (iPSCs) originated from GM1 gangliosidosis patient and by constructing the differentiated tissue-specific cells derived from the iPSCs, and a use of the said cell model for the screening of a GM1 gangliosidosis treating agent.
2. Description of the Related Art
GM1 gangliosidosis (GM1) is a very rare hereditary disease caused by deficiency of lysosomal β-galactosidase β-gal), which is thus classified into lysosomal storage disease (LSD). β-gal is an enzyme encoded by GLB1 gene existing in lysosome, which plays a role in hydrolysis of various biomolecules (Brunetti-Pierri N and Scaglia F, molecular genetics and metabolism 94, 391-396, 2008). The most representative substrate of β-gal is GM1 ganglioside, the lysosomal sphingolipid. GM1 ganglioside is rich in the brain and plays an important role in the development and the general functions of nerve cells (Yu R K et. al, Neurochemical research 37, 1230-1244, 2012). The deficiency of β-gal activity leads to the accumulation of GM1 ganglioside in other cellular organs including endoplasmic reticulum (ER), and accordingly causes various symptoms including seizures, ataxia, and hepatosplenomegaly. The severity of such symptoms and the point of outbreak of disease are presumed to be related to the remaining β-gal activity. The most peculiar clinical symptom of GM1 is the progressive neurodegeneration in CNS. Thus, GM1 is basically understood as a neurological disorder and seems to share very similar characteristics with other neurodegenerative diseases (Vitner E B et. al, The Journal of biological chemistry 285, 20423-20427, 2010; Sandhoff K and Harzer K. The Journal of neuroscience: the official journal of the Society for Neuroscience 33, 10195-10208, 2013).
In the study on the developmental mechanism of GM1, the β-gal knock-out mouse model has been used. From which, various mechanisms responding to neuronal cell death such as unfolded protein response (UPR), mitochondrial dysfunction, increased autophagy, and activation of Trk signaling have been proposed (Tessitore A, et al. molecular cell 15, 753-766, 2004; Sano R, et al. molecular cell 36, 500-511, 2009; Takamura A, et al. Biochemical and biophysical research communications 367, 616-622, 2008; Takamura A, et al. Journal of neurochemistry 118, 399-406, 2011). The activation of inflammatory factor was once confirmed in the brain of β-gal−/− mouse. The progressive CNS inflammation is closely related to the clinical symptoms caused by such activation (Jeyakumar M, et al. Brain: a journal of neurology 126, 974-987, 2003). Chronic inflammation in CNS has been included in the criteria of neurodegenerative disease in a wide sense, and has been known to make abnormality in the brain structure and brain functions worse. Nevertheless, the molecular mechanism of the chronic neuroinflammation has not been fully studied.
The new role of inflammasome has recently confirmed in chronic inflammation related disease (Walsh J G et. al, Nature reviews Neuroscience 15, 84-97, 2014; Choi A M and Nakahira K, Nature immunology 12, 379-380, 2011; Franchi L et. al, Nature immunology 10, 241-247, 2009; Kufer T A and Sansonetti P J, Nature immunology 12, 121-128, 2011). According to the recent report, the said inflammasome is a protein complex composed of three kinds of proteins such as sensor protein, adaptor protein, and caspase-1. Various stimuli caused by infection, cell damage, and intracellular stress accelerate the formation of inflammasome and promote the activation of caspase-1. The activated caspase-1 induces the maturation of IL1β, the proinflammatory cytokine, and produces cytokine by secreting IL1β thereafter, resulting in the inducement of chronic inflammation. Therefore, inflammasome has been studied with immune cells including macrophages and microglias. Also, studies have been going on to prove any function of inflammasome in CNS neurons (Walsh J G et. al, Nature reviews Neuroscience 15, 84-97, 2014). More importantly, the activation of inflammasome has been explained as a molecular mechanism of the development of neurodegenerative disease including Alzheimer's disease and Parkinson's disease (Heneka M T, et al. Nature 493, 674-678, 2013). Amyloid-β well-known as the molecule that causes Alzheimer's disease induces the activation of inflammasome by destroying lysosome (Halle A, et al. Nature immunology 9, 857-865, 2008). Fibrillar α-synuclein playing an important role in causing Parkinson's disease can increase the production of ROS and make lysosome unstable to finally activate inflammasome (Codolo G, et al. PloS one8, e55375, 2013).
The molecular mechanism related to human GM1 development has not been fully disclosed so far, so that the treatment of GM1 is still not possible. Even though GM1 mouse model has been constructed and this model can emulate many characteristics of human GM1, it can never be replaced with human GM1 and therefore study with GM1 mouse model will soon reach the limit. Therefore, the development of a human originated GM1 cell model that can reproduce the GM1 development mechanism will be very useful to understand the mechanism and cause of the disease and also to establish a more efficient treatment method.
Stem cells are the cells in the phase of pre-differentiation before being differentiated into each tissue forming cells, which can be obtained from the tissues of an embryo, a fetus, and an adult. Stem cells have self-proliferative activity that makes unlimited proliferation possible from undifferentiated status and have pluripotency, so that they can be differentiated into various tissue cells once a certain stimulus is given. That is, stem cells become to be differentiated by a certain differentiation stimulus (environment), and are self-renewal so as to produce the cells that are same as themselves by cell division, unlike the differentiated cells whose cell division has been finished. Stem cells also have proliferation/expansion capacity and plasticity, by which stem cells can be differentiated into different cells when the environment is changed or when a different stimulus is given.
Human pluripotent stem cells (hPSCs) including induced pluripotent stem cells (iPSCs) have excellent differentiation potency, so that they can be differentiated into almost every tissue cells forming human body. In particular, patient-originated iPSCs can produce tissue-specific differentiated cells showing immunologically and genetically same characteristics as the patient's, in the in vitro differentiation system. Thus, human pluripotent stem cells are well-known as the effective evaluator not only for the development of patient-customized cell therapy products which are free from worry on immune rejection response but also for understanding complicated disease mechanism in the early stage of organogenesis (Muotri, A. R. (2009) Epilepsy Behav 14 Suppl 1: 81-85; Marchetto, M. C., B. Winner, et al. (2010) Hum Mol Genet 19(R1): R71-76).
It was reported previously that when patient originated iPSCs obtained from patients with various genetic diseases were directly differentiated into disease-related cells, disease-specific phenotypes were observed (Park, I. H. et al. Cell 134, 877-886 (2008); Tiscornia, G. et al. Nature medicine 17, 1570-1576 (2011)). Such disease-specific iPSCs can be differentiated into those tissues that are directly involved in the cause of a disease or cell damage. So, the differentiated tissue-cell displaying disease specific characteristics can be used for the study on the mechanism to explain a cause of disease or for the development of a treating agent of the disease.
The present inventors tried to establish a human cell model for the study of GM1 gangliosidosis (GM1) based on patient-originated iPSCs. As a result, the inventors first constructed GM1 originated induced pluripotent stem cells (iPSCs) from fibroblasts of GM1 patient and then induced the differentiation of embryoid body (EB) and neural progenitor cells (NPCs) from the same. The GM1 patient derived iPSCs display both in vitro and in vivo pluripotency and at the same time have GM1 causing gene mutation detected in GM1 patient and accordingly show the reduced β-gal activity. The inventors also induced the differentiation of iPSCs originated from GM1 patient into neural progenitor cells. As a result, the expression of β-gal was increased but the activity thereof was reduced in the differentiated neural progenitor cells, suggesting that intracellular GM1 ganglioside and lysosome accumulation was increased. The gene expression pattern in the GM1 originated neural progenitor cells was compared with that of the normal cell. As a result, it was confirmed that inflammation related pathway, particularly inflammasome related metabolic pathway, was promoted. When the neural progenitor cells differentiated from GM1 patient derived iPSCs were treated with an inflammasome inhibitor, not only cell morphology and size but also gene expression pattern were recovered similarly to those of normal cells. The above results suggest that GM1 outbreak is related to inflammasome activation and therefore the inhibition of inflammasome is functioning to treat GM1. The present inventors examined disease-specific phenotype by using GM1 patient originated induced pluripotent stem cells (iPSCs) and iPSCs derived neural progenitor cells. As a result, it was suggested that inflammasome can be a key molecular target of the study to develop a GM1 treating agent and an inflammasome inhibitor displays positive effect on GM1 treatment. In conclusion, the present inventors completed this invention by proposing a novel GM1 gangliosidosis human cell model that can be efficiently used for the study of cause of GM1 and for the development of a therapeutic agent for the disease.